The heptad repeat region 2 (HR2) of HIV-1 gp41 protein is involved in the formation of a coiled-coil structure with an N-terminal heptad repeat region on gp41 (HR1) that plays an important role in fusion. Binding of gp120 to the CD4 receptor results in a conformational rearrangement that is believed to expose portions of gp41 and initiate its rearrangement into a six helix bundle. An important role for this rearrangement in infection is indicated by the potent inhibition of infection by a large helical peptide derived from the HR2 domain that binds to a portion of HR1 and prevents the six helix bundle formation. This peptide, marketed as the antiviral drug FUZEON®, is composed of amino acid residues CH3CO-Tyr-Thr-Ser-Leu-Ile-His-Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2 (SEQ ID NO:1).
Neutralization is the ability of antibody to bind to and inactivate virus infectivity. Neutralization by antibody can be mediated by a number of different mechanisms including aggregation of virions, destabilization of the virion structure, inhibition of virion attachment to target cells, inhibition of the fusion of the virion lipid membrane with the membrane of the host cell, inhibition of the entry of the genome of non-enveloped viruses into the cell cytoplasm, blocking of protein rearrangements needed for one of these functions, inhibition of a function of the virion core through a signal transduced by an antibody, and binding to nascent virions to block their budding or release from the cell surface. A virus may be neutralized by several different mechanisms because several unique epitopes may be sited in different locations on the virion and paratope and other properties of the reacting antibody may vary.
A variety of epitopes for HIV-1 binding antibodies are known in the art. Such epitopes are found in gp120, and gp41. See, for example, HIV Molecular Immunology (2002) Korber et al. ed., Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N. Mex. LA-DR 03-5816. Although many antibody binding sites have been described in gp41, only a few lead to neutralization. For example, three monoclonal antibodies isolated from infected humans, 2F5, 4E10 and Z13, have been shown to recognize sequences in the membrane proximal external region (MPER) of gp41. The epitopes recognized by these monoclonal antibodies possess the attractive features that they are highly conserved and mediate relatively potent neutralization of virions, including viruses that are resistant to standard antibodies that target the major sites on gp120. The predominant disadvantage of these epitopes is that they are poorly immunogenic.
Recent evidence suggests a key property of the MPER-specific monoclonal antibodies is recognition of lipid components of the adjacent membrane in addition to binding to a peptide determinant on gp41 (Alam, et al. (2007) J. Immunol. 178:4424-4435). As a result, these antibodies also bind to normal cellular lipid components, including cardiolipin, and thus possess some autoimmune properties. It has been suggested that this autoreactive property accounts for the inability of most individuals to produce similar antibodies with HIV-neutralizing properties.
A series of nineteen monoclonal antibodies and Fab fragments have been described that have been mapped to gp41 “Cluster II”, defined by reactivity with a fusion protein that contains the sequence Ser-Leu-Ile-Glu-Glu-Ser-Gln-Asn-Gln-Gln-Glu-Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu (SEQ ID NO:2), corresponding to gp41 residues 644-663 (HIV Immunology and HIV/SIV Vaccine Database 2003). However, all of the monoclonal antibodies mapped to this region have been described as “non-neutralizing” (Gorny and Zolla-Pazner (2003) In HIV Immunology and HIV/SIV Vaccine Databases. Korber, et al., editors. Publisher: Los Alamos National Laboratory, Theoretical Biology and Biophysics, Los Alamos, N. Mex. LA-UR 04-8162. pp. 37-51). In addition, six gp41-specific Fabs have been assigned to “Cluster III”, defined as a conformational epitope involving amino acid residues 619-648, and these too were characterized as non-neutralizing. The lack of neutralizing activity of the previously described monoclonal antibodies and Fabs suggests that these reagents would not be useful in the neutralization of HIV and that therefore this region would not be a useful vaccine target.
Similarly, WO 2005/111621 suggests epitopes of gp41 including the disulfide-loop region of gp41 that links the N-HR and C-HR regions (amino acid residues 581 to 628), the N-HR region of gp41 (amino acid residues 546 to 581), the C-HR of gp41 (amino acid residues 628 to 661), and the membrane proximal region of gp41 (amino acid residues 657 to amino acids 684). However, neutralizing antibodies to each region are not provided.